Response of the Ubiquitous Pelagic Diatom Thalassiosira weissflogii to Darkness and Anoxia

Thalassiosira weissflogii, an abundant, nitrate-storing, bloom-forming diatom in the world’s oceans, can use its intracellular nitrate pool for dissimilatory nitrate reduction to ammonium (DNRA) after sudden shifts to darkness and anoxia, most likely as a survival mechanism. T. weissflogii cells that stored 4 mM ¹⁵N-nitrate consumed 1.15 (±0.25) fmol NO₃ ^- cell^-1 h^-1 and simultaneously produced 1.57 (±0.21) fmol ¹⁵NH₄ ⁺ cell^-1 h^-1 during the first 2 hours of dark/anoxic conditions. Ammonium produced from intracellular nitrate was excreted by the cells, indicating a dissimilatory rather than assimilatory pathway. Nitrite and the greenhouse gas nitrous oxide were produced at rates 2-3 orders of magnitude lower than the ammonium production rate. While DNRA activity was restricted to the first few hours of darkness and anoxia, the subsequent degradation of photopigments took weeks to months, supporting the earlier finding that diatoms resume photosynthesis even after extended exposure to darkness and anoxia. Considering the high global abundance of T. weissflogii, its production of ammonium and nitrous oxide might be of ecological importance for oceanic oxygen minimum zones and the atmosphere, respectively.     

Effect of Exogenous Glucose on Photosynthesis in the Diatom Thalassiosira weissflogii Depending on Nitrate Nitrogen Supply and Illumination

The rate of photosynthetic carbon fixation (P) in the diatom Thalassiosira weissflogii cultivated in the presence of exogenous glucose in the medium (0–10.56 g C/l) at different levels of illumination—25, 50, and 100 E/(m² s)—was studied as a function of nitrate nitrogen supply. In the diatoms limited in nitrogen and assimilating exogenous glucose, P was found to decrease or increase depending on the light intensity, glucose concentration, and the duration of exposure. In the diatoms assimilating both nitrate nitrogen and glucose, compared to those supplied with nitrates alone, P was higher at the medium and high light intensities and lower at the low light intensity. The interrelation of the processes of carbon and nitrogen metabolism in mixotrophic algae and the ecological role of glucose uptake by phytoplankton are discussed.     

Identification of a New Gene Family Expressed during the Onset of Sexual Reproduction in the Centric Diatom Thalassiosira weissflogii

An intriguing feature of the diatom life cycle is that sexual reproduction and the generation of genetic diversity are coupled to the control of cell size. A PCR-based cDNA subtraction technique was used to identify genes that are expressed as small cells of the centric diatom Thalassiosira weissflogii initiate gametogenesis. Ten genes that are up-regulated during the early stages of sexual reproduction have been identified thus far. Three of the sexually induced genes, Sig1, Sig2, and Sig3, were sequenced to completion and are members of a novel gene family. The three polypeptides encoded by these genes possess different molecular masses and charges but display many features in common: they share five highly conserved domains; they each contain three or more cysteine-rich epithelial growth factor (EGF)-like repeats; and they each display homology to the EGF-like region of the vertebrate extracellular matrix glycoprotein tenascin X. Interestingly, the five conserved domains appear in the same order in each polypeptide but are separated by variable numbers of nonconserved amino acids. SIG1 and SIG2 display putative regulatory domains within the nonconserved regions. A calcium-binding, EF-hand motif is found in SIG1, and an ATP/GTP binding motif is present in SIG2. The striking similarity between the SIG polypeptides and extracellular matrix components commonly involved in cell-cell interactions suggests that the SIG polypeptides may play a role in sperm-egg recognition. The SIG polypeptides are thus important molecular targets for determining when and where sexual reproduction occurs in the field.     

The influence of cell size on the growth rate of Thalassiosira weissflogii

Growth rate and average cell volume were measured throughoutauxospore formation in two populations of Thalassiosira weissflogii(Hustedt). In both cases, the entire population shifted fromrelatively small (800 µm³) to large cells (2800 µm³)over a 5 day interval. This shift was accompanied by a dramaticincrease in the average growth rate of the populations from1.6 to 3.4 doublings/day.     

Effects of Nutrient Limitation on Pigments in Thalassiosira weissflogii and Prorocentrum donghaiense

The response of Prorocentrum donghaiense and Thalassiosira weissfiogii pigments under nitrate （N） and phosphate （P） limitation were studied using HPLC and in vivo fluorescence protocols in batch cultures. For P. donghaiense, the pigment ratio was kept stable under different nutrient conditions from the results of HPLC. For T. weissflogii, there was a lower ratio of chlorophyllide to Chl a during the exponential phase, but the reverse during the stationary phase. Different members of the phytoplankton had different pigments response mechanisms under nutrient limitation. From the results of in vivo fluorescence, the ratio of peridinin to Chl a for P. donghaiense increased in nutrient-free culture, while it was kept stable for nutrient-limited cultures during the exponential phase. For T. weissflogii, the ratio of fucoxanthin to Chl a for each culture increased during the exponential phase, but the ratio under N limitation was apparently lower than that for P limitation during the stationary phase. The results indicate that both pigment ratios from HPLC and in vivo fluorescence of To weissflogii were changed greatly under different nutrient conditions, which suggests that both ratios could be used as indicators of algal physiological status in different nutrient conditions.     

Growth and photosynthetic activity of diatom Thalassiosira weissflogii at decreasing salinity

The relative yield of variable chlorophyll fluorescence (F _v/F _m), rate of photosynthetic carbon fixation (P), growth rate, and production of extracellular photosynthates (P _ec) was studied in diatom T. weissflogii in seawater with salinity decreasing from 35 to 15 and 5‰. After incubation at 5‰ for 1 day, the diatom abundance (N) decreased as a result of death of a fraction of cells, while viable cells demonstrated decreased F _v/F _m and P, P _ec was detectable, and cell division was likely inhibited. After incubation for 2 days, population started to grow, while exponential growth rate and the abundance by day 8 were lower compared to 35‰. After incubation at 15‰ for 1 day, P was higher and F _v/F _m was lower compared to 35‰. No cell death was observed and exponential growth rate insignificantly differed from that at 5‰. The value of N by day 8 was lower compared to 35‰ but higher compared to 5‰. The dependence of photosynthetic parameters and population dynamics of T. weissflogii on the relative salinity is discussed.     

Cell cycle implication on nitrogen acquisition and synchronization in Thalassiosira weissflogii (Bacillariophyceae)

The Michaelis–Menten model of nitrogen (N) acquisition, originally used to represent the effect of nutrient concentration on the phytoplankton uptake rate, is inadequate when other factors show temporal variations. Literature generally links diurnal oscillations of N acquisition to a response of the physiological status of microalgae to photon flux density (PFD) and substrate availability. This work describes how the cell cycle also constitutes a significant determinant of N acquisition and, when appropriate, assesses the impact of this property at the macroscopic level. For this purpose, we carried out continuous culture experiments with the diatom Thalassiosira weissflogii (Grunow) G. Fryxell & Hasle exposed to various conditions of light and N supply. The results revealed that a decrease in N acquisition occurred when a significant proportion of the population was in mitosis. This observation suggests that N acquisition is incompatible with mitosis and therefore that its acquisition rate is not constant during the cell cycle. In addition, environmental conditions, such as light and nutrient supply disrupt the cell cycle at the level of the individual cell, which impacts synchrony of the population.     

Purification and Characterization of the Nitrate Reductase from the Diatom Thalassiosira pseudonana

The assimilatory nitrate reductase (NADH: nitrate oxidoreductase, E.C. 1.6.6.2.) from the marine diatom Thalassiosira pseudonana, Hasle and Heimdal, has been purified 200-fold and characterized. The regulation of nitrate reductase in response to various conditions of nitrogen nutrition has been investigated.     

Effect of mercuric chloride and methylmercury on photosynthetic activity of the diatom Thalassiosira weissflogii by a fluorescent method

It was shown by the pulse-amplitude modulation fluorescent method that, at a weak illumination (6 microE m-2.s-1), methylmercury at a concentration of 10(-6)-10(-7) M decreases the photochemical activity of the reaction centers of photosystem II in cells of microalgae Thalassiosira weissflogii after a prolonged lag phase. Cells resistant to methylmercury at these low concentrations were detected by the microfluorimetric method. Chloride mercury decreased the activity of photosystem II of the algae only when at higher concentrations. Both toxicants at a concentration of 10(-6) M decreased the rate of recovery of photoinduced damage of centers of photosystem II and led to an increase in the energization component of nonphotochemical fluorescence quenching. These results indicate that the complex of fluorescent methods can be used to monitor early changes in the photosynthetic apparatus of algae in response to the toxic action of heavy metals.     

Comparative phosphorus nutrition of the marine cyanobacterium Synechococcus WH7803 and the marine diatom Thalassiosira weissflogii

Phosphate uptake kinetics of Synechococcus sp. WH7803 and Thalassiosiraweissflogii were studied in axenic batch culture. Phosphate-repleteSynechococcus sp. WH7803 cells have a lower affinity for inorganicphosphate (Pi) (K_s = 67 µmol l^–1) than Pi-starvedcells (K_s = 3.1 µmol l^–1). The K_s of Pi-starvedcells increased     

Effects of Detergents on in vitro Growth of Diatom Alga Thalassiosira pseudonana

The growth of the diatom alga Thalassiosira pseudonana was studied when exposed to an environment polluted by a detergent. We determined concentrations that inhibit cell division (10 mg/l) instead of algae growth (0.1 and 1 mg/l. It was shown that T. pseudonana can adapt to high detergent concentrations. The stimulation of the growth of Thalassiosira within a range of 0.03–0.08 mg/l concentration has been registered.     

Structural and inhibition insights into carbonic anhydrase CDCA1 from the marine diatom Thalassiosira weissflogii

Carbonic anhydrases (CAs) catalyze with high efficiency the reversible hydration of carbon dioxide, an essential reaction for many biological processes, such as photosynthesis, respiration, renal tubular acidification, and bone resorption. Diatoms, which are one of the most common types of phytoplankton and are widespread in oceans, possess CAs fundamental for acquisition of inorganic carbon. Recently, in the marine diatom Thalassiosira weissflogii a novel enzyme, CDCA1, naturally using Cd in its active site, has been isolated and categorized in a new CA class, namely zeta-CA. This enzyme, which consists of three repeats (R1, R2 and R3), is a cambialistic carbonic anhydrase that can spontaneously exchange Zn or Cd at its active centre, presumably an adaptative advantage for diatoms that grow fast in the metal-poor environment of the surface ocean. In this paper we completed the characterization of this enzyme, reporting the X-ray structure of the last repeat, CDCA1-R3 in its cadmium-bound form, and presenting a model of the full length protein obtained by docking approaches. Results show that CDCA1 has a quite compact not symmetric structure, characterized by two covalently linked R1-R2 and R2-R3 interfaces and a small non-covalent R1-R3 interface. The three dimensional arrangement shows that most of the non-conserved aminoacids of the three repeats are located at the interface regions and that the active sites are far from each other and completely accessible to the substrate. Finally, a detailed inhibition study of CDCA1-R3 repeat in both cadmium- and zinc- bound form has been performed with sulfonamides and sulfamates derivatives. The results have been compared with those previously reported for other CA classes, namely alpha- and beta-classes, and correlated with the structural features of these enzymes.     

Effect of Si-status on diel variation of intracellular free amino acids in Thalassiosira weissflogii under low-light intensity

Twenty-one intracellular free amino acids were analysed during a 12-12 h light-dark cycle, on duplicate axenic cultures of Thalassiosira weissflogii (clone Actin, Provasoli-Guillard CCMP) under either Si-sufficient or Si-starved conditions. Total concentrations ranged between 40 and 165 fmol/cell. Total level as well as individual levels of amino acids decreased during the dark period, and GLN/GLU ratio was lower during the dark period. All these results were correlated with the light-dark carbon metabolism of the algae and related to the protein synthesis at night. The Si-starved cultures showed a lower total level of FAA compare to the Si-sufficient cultures, especially in the light period. Silica status of the cells affected more the metabolites of the dark respiration than the photorespiratory metabolites SER and GLY. Si deprivation induced higher range of ALA and VAL, and a decrease of the TCA metabolites GLU & ASP. Additionally, the relative percentage of ASP increased under Si starvation, at the expense of GLU, and this shift was emphasized in the dark period.     

Grazing rates of Calanus finmarchicus on Thalassiosira weissflogii cultured under different levels of ultraviolet radiation

UVB alters photosynthetic rate, fatty acid profiles and morphological characteristics of phytoplankton. Copepods, important grazers of primary production, select algal cells based upon their size, morphological traits, nutritional status, and motility. We investigated the grazing rates of the copepod Calanus finmarchicus on the diatom Thalassiosira weissflogii cultured under 3 levels of ultraviolet radiation (UVR): photosynthetically active radiation (PAR) only (4 kJ-m(-2)/day), and PAR supplemented with UVR radiation at two intensities (24 kJ-m(-2)/day and 48 kJ-m(-2)/day). There was no significant difference in grazing rates between the PAR only treatment and the lower UVR treatment. However, grazing rates were significantly (～66%) higher for copepods feeding on cells treated with the higher level of UVR. These results suggest that a short-term increase in UVR exposure results in a significant increase in the grazing rate of copepods and, thereby, potentially alters the flow rate of organic matter through this component of the ecosystem.     

Characterization of an Endoplasmic Reticulum-associated Silaffin Kinase from the Diatom Thalassiosira pseudonana

The formation of SiO₂-based cell walls by diatoms (a large group of unicellular microalgae) is a well established model system for the study of molecular mechanisms of biological mineral morphogenesis (biomineralization). Diatom biomineralization involves highly phosphorylated proteins (silaffins and silacidins), analogous to other biomineralization systems, which also depend on diverse sets of phosphoproteins (e.g. mammalian teeth and bone, mollusk shells, and sponge silica). The phosphate moieties on biomineralization proteins play an essential role in mineral formation, yet the kinases catalyzing the phosphorylation of these proteins have remained poorly characterized. Recent functional genomics studies on the diatom Thalassiosira pseudonana have revealed >100 proteins potentially involved in diatom silica formation. Here we have characterized the biochemical properties and biological function of one of these proteins, tpSTK1. Multiple tpSTK1-like proteins are encoded in diatom genomes, all of which exhibit low but significant sequence similarity to kinases from other organisms. We show that tpSTK1 has serine/threonine kinase activity capable of phosphorylating silaffins but not silacidins. Cell biological and biochemical analysis demonstrated that tpSTK1 is an abundant component of the lumen of the endoplasmic reticulum. The present study provides the first molecular structure of a kinase that appears to catalyze phosphorylation of biomineral forming proteins in vivo.     

Effects of fundamental nutrient stresses on the lipid accumulation profiles in two diatom species Thalassiosira weissflogii and Chaetoceros muelleri

Bioprocess and Biosystems Engineering - Microalgae are considered as attractive feedstocks for biofuel production nowadays because of their high lipid contents and easy cultivation. In the present...     

Biochemical Composition and Assembly of Biosilica-associated Insoluble Organic Matrices from the Diatom Thalassiosira pseudonana

The nano- and micropatterned biosilica cell walls of diatoms are remarkable examples of biological morphogenesis and possess highly interesting material properties. Only recently has it been demonstrated that biosilica-associated organic structures with specific nanopatterns (termed insoluble organic matrices) are general components of diatom biosilica. The model diatom Thalassiosira pseudonana contains three types of insoluble organic matrices: chitin meshworks, organic microrings, and organic microplates, the latter being described in the present study for the first time. To date, little is known about the molecular composition, intracellular assembly, and biological functions of organic matrices. Here we have performed structural and functional analyses of the organic microrings and organic microplates from T. pseudonana. Proteomics analysis yielded seven proteins of unknown function (termed SiMat proteins) together with five known silica biomineralization proteins (four cingulins and one silaffin). The location of SiMat1-GFP in the insoluble organic microrings and the similarity of tyrosine- and lysine-rich functional domains identifies this protein as a new member of the cingulin protein family. Mass spectrometric analysis indicates that most of the lysine residues of cingulins and the other insoluble organic matrix proteins are post-translationally modified by short polyamine groups, which are known to enhance the silica formation activity of proteins. Studies with recombinant cingulins (rCinY2 and rCinW2) demonstrate that acidic conditions (pH 5.5) trigger the assembly of mixed cingulin aggregates that have silica formation activity. Our results suggest an important role for cingulins in the biogenesis of organic microrings and support the hypothesis that this type of insoluble organic matrix functions in biosilica morphogenesis.     

Aggregation and Sedimentation of Thalassiosira weissflogii (diatom) in a Warmer and More Acidified Future Ocean

Increasing Transparent Exopolymer Particle (TEP) formation during diatom blooms as a result of elevated temperature and pCO₂ have been suggested to result in enhanced aggregation and carbon flux, therewith potentially increasing the sequestration of carbon by the ocean. We present experimental results on TEP and aggregate formation by Thalassiosira weissflogii (diatom) in the presence or absence of bacteria under two temperature and three pCO₂ scenarios. During the aggregation phase of the experiment TEP formation was elevated at the higher temperature (20°C vs. 15°C), as predicted. However, in contrast to expectations based on the established relationship between TEP and aggregation, aggregation rates and sinking velocity of aggregates were depressed in warmer treatments, especially under ocean acidification conditions. If our experimental findings can be extrapolated to natural conditions, they would imply a reduction in carbon flux and potentially reduced carbon sequestration after diatom blooms in the future ocean.     

Direct impact of silicate on the photosynthetic performance of the diatom Thalassiosira weissflogii assessed by on- and off-line PAM fluorescence measurements

Pulse-amplitude-modulation (PAM) fluorometry was used to investigate the effects of varying the silicate concentration on different fluorescence characteristics of batch and chemostat cultures of the diatom Thalassiosira weissflogii. The fluorescence signals, measured both on- and off-line, were used to calculate the actual (P) and potential (P0) photochemical efficiencies of the reaction centres of photosystem II (PSII). Also the relative electron transport rate of the reaction centres of PSII (Je) was calculated. Fluorescence decreased in silicate-limited cells and increased rapidly after silicate addition. The decrease was caused by strong non-photochemical quenching (qN) in silicate-limited cells. Continuous recording (on-line) of the minimum and maximum fluorescence provided data with high temporal resolution, revealing that the first recovery of silicate-limited cells occurred 20 min after the addition of silicate. Based on these observations, we assume a strong influence of silicate metabolism on the photosynthetic efficiency of the reaction centres of PSII assessed by PAM fluorescence. This implies that silicate, as well as other nutrients, has to be considered as a possible cause for variable photosynthetic efficiency of diatoms.